Terminally differentiated, incapable of dividing, the sensory hair cells of the mammalian auditory organ must last a lifetime. This challenge, daunting in any tissue, is further complicated in the organ of Corti (OC) by the absence of vasculature, a highly positive electrical potential, and the continuous "silent" K+ current. In this setting, even transient departures from the physiological norm result in hair cell death and consequent hearing loss. Ironically, the gene most commonly linked to hereditary deafness disorders (connexin 26, Cx26) is not expressed in the sensory cell, but rather in the supporting cell population, or epithelial support complex (ESC). Two common features unite the diverse cell types of the ESC - an elaborate epithelial gap-junction system (EGJS) and high-level expression of OCP1 and OCP2. Formed by hexagonal arrays of connexin molecules, gap-junctions facilitate movement of low molecular weight compounds - nutrients, metabolites, signaling molecules - between adjacent cells. As the severity of the Cx26 phenotype illustrates, disruption of this transport system has disastrous implications for sensory cell survival. Compelling preliminary evidence links OCP1 and OCP2 to regulation of the EGJS. The two most abundant proteins in the OC, OCP1 and OCP2 display homology to subunits of SCF E3 ubiquitin ligases. These multi-component, multi-functional complexes regulate a broad spectrum of biological processes, typically by targeting critical proteins for ubiquitination. Depending on the precise molecular details of the ubiquitin signal, the modified protein will experience one of several potential fates, including proteolytic destruction or sub-cellular relocation. The coincidence of OCP1-OCP2 expression with the boundaries of the EGJS suggests that the OCP1-OCP2 complex may participate in the ubiquitination of one or more connexin isoforms. This hypothesis is bolstered by the recent demonstration that OCP1 binds to Cx2.6 in vitro. The objective of the proposed research is to examine the functional significance of the OCP1-OCP2 complex and, in particular, its interrelationship with the EGJS. The multi-disciplinary project includes targeted disruption of the OCP1 gene, identification of additional OCP1 target proteins, and biochemical/biophysical characterization of the OCP1-OCP2 complex and OCPl-target complexes. This work will furnish insight into the physiological processes underlying preservation of hair cell function. It will advance our understanding of the system (Cx26) most commonly responsible for congenital hereditary hearing loss. Ultimately, the data from this project could provide a foundation for genetic and pharmacologic intervention in hearing disorders.